Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease and of preventable blindness in humans. Antibiotics (not inexpensive) are used to control ongoing infections but common re-infection frequently causes serious pathology, e.g. pelvic inflammatory disease, infertility and blindness. Despite growing evidence of versatile human B cell and T cell immune responses to Ct antigens, there is little evidence of long-lasting protective immunity following infection; somehow, the organism evades repulse or elimination by the immune system. Current inability to genetically manipulate Ct has impeded analysis that might increase understanding of how Ct infections work and how the immune system might be better engaged in the management of Ct infection. The proposed work is aimed at developing a method of introducing planned genetic modifications into many targeted Ct genes following a three step work plan: (i) Demonstrate how to genetically transform Ct by means of homologous recombination between cloned Ct DNA that is transferred into Ct and chromosomal DNA of recipient Ct. A cloned mutant gyr A gene that renders Ct resistant to ofloxacin (OFX) will be transferred into sensitive Ct and resistant transformants will be isolated by selection with OFX. (ii) Demonstrate how to use the results of (i) to replace a normal Ct gene with a cloned mutant allele by the use of a model 'homologous recombination vector' (HRV). The same mutant gyr A gene used for (i) will be used, but transformants will be isolated by selection for a different, non Ct- derived 'selection marker' that is part of the HRV. Homologus recombination in Ct-derived parts of the HRV will incorporate the selection marker and closely linked Ct DNA into the Ct chromosome, thereby replacing the indigenous gene with the mutant trans-gene. (iii) Use the results of (ii) to create model knockout mutant Ct strains that can be studied in animal models. A knockout mutant allele of the folA gene will be used for this model because folA - deficient transformants that normally would be unviable can be isolated by supplementation of the culture medium with reduced folic acid. A multitude of mutation/function investigations that could be based on these model demonstrations includes the possible development of attenuated strains of Ct that might be useful protective vaccines. Pairs of mutants used in (i) - (iii) above will also be used to detect genetic recombination in mixedly infected human host cells. There is clinical evidence that such recombination occurs in humans and may contribute to Ct evasion of protective immune responses.